Denervation activated super-enhancers of pathogenic IL6-STAT3 feedforward loop in FAPs

项目摘要 该提案研究了致病性IL 6-STAT 3的组成性激活的表观遗传机制， 来自失神经肌肉的纤维脂肪生成祖细胞（FAP）中的信号传导（DEN FAP）。FAP至关重要 骨骼肌适应环境扰动能力的细胞效应器。为了应对急性损伤， FAP在炎性细胞和卫星细胞（SC）的协调激活中被激活，并有助于 促进肌肉修复。相比之下，在对慢性损伤（例如肌营养不良）的反应中， FAP、巨噬细胞（MP）和SC之间的功能相互作用导致再生受损， 纤维化和脂肪沉积导致的适应不良修复。我们最近发现了FAP的另一种模式 激活响应肌肉去神经支配，从而在没有伴随的MP浸润和 SC激活，DEN FAP的进行性扩增以及IL 6-STAT 3信号传导的持续激活导致 肌纤维萎缩和纤维化。我们认为，灵活使用基因组的顺式调控元件， FAP采用不同的功能表型来响应不同类型的骨骼肌扰动， 通过激活特定的基因表达模式。在这方面，IL 6的差异活化 在FAP对不同类型的扰动（即急性损伤与去神经支配）的反应期间观察到的转录 为研究顺式调控元件之间的动态相互作用如何调节 阈值，幅度和持续时间的基因转录响应不同的刺激。我们将调查 假设在DEN FAPs中，STAT 3促进远端增强子和高度依赖性转录因子之间的长程相互作用。 IL-6转录起始位点（TSS）附近的保守调控元件，以激活组成型高表达 IL 6转录和分泌水平，这反过来又通过自分泌驱动STAT 3的长期激活， IL 6受体的结合。由于FAP是由动态的离散子群体（subFAP）组成的， 平衡，我们假设IL 6-STAT 3信号传导的组成性激活导致了一种自我调节的出现。 自治，占主导地位的亚群，占登FAP的致病活动。具体 目的是检验STAT 3介导的IL 6超增强子的激活是IL-6表达的关键事件这一假设。 建立致病性前馈IL 6-STAT 3环。在目标1（DEN FAP中IL 6基因座的3D调节）中， 我们将使用基于染色体构象捕获（3C）和ChIP的技术来识别和表征 DEN FAP中的IL 6基因相互作用增强子。在目标2（检测DEN子FAP及其 功能表征），我们将利用单细胞RNAseq来检测DEN subFAP并建立一个 IL 6-STAT 3超活化与其自我自主扩增之间的功能关系。在目标3 在DEN FAP中STAT 3激活的超级增强子的药理学靶向，我们将使用 STAT 3激活的超级增强子的药理学抑制，以确定IL 6- STAT 3前馈回路防止DEN subFAP的致病潜力。

PROJECT SUMMARY This proposal investigates the epigenetic mechanism of constitutive activation of pathogenic IL6-STAT3 signaling in Fibro-Adipogenic Progenitors (FAPs) from denervated muscles (DEN FAPs). FAPs are essential cellular effectors of skeletal muscle ability to adapt to environmental perturbations. In response to acute injury, FAPs are activated within a coordinate activation of inflammatory and satellite cells (SCs), and contribute to promote muscle repair. By contrast, in response to chronic injury (e.g. muscular dystrophies) dysregulated functional interactions between FAPs, macrophages (MPs) and SCs lead to impaired regeneration and maladaptive repair by fibrosis and fat deposition. We have recently discovered an alternative pattern of FAPs activation in response to muscle denervation, whereby in the absence of concomitant infiltration of MPs and SC activation, a progressive expansion of DEN FAPs with persistent activation of IL6-STAT3 signaling leads to myofiber atrophy and fibrosis. We posit that a flexible usage of cis-regulatory elements of the genome enables FAPs to adopt different functional phenotypes in response to distinct types of skeletal muscle perturbations, through activation of specific patterns of gene expression. In this regard, the differential activation of IL6 transcription observed during FAP’s response to different types of perturbation (i.e. acute injury vs denervation) provides an opportunity to investigate how dynamic interactions between cis-regulatory elements regulate threshold, magnitude and duration of gene transcription in response to distinct stimuli. We will investigate the hypothesis that in DEN FAPs STAT3 promotes long-range interactions between distal enhancers and a highly conserved regulatory element proximal to the IL6 transcription start site (TSS), to activate constitutive high levels of IL6 transcription and secretion, which in turn drives prolonged activation of STAT3, via autocrine engagement of IL6 receptor. As FAPs are composed of discrete sub-populations (subFAPs) in dynamic equilibrium, we hypothesize the constitutive activation of IL6-STAT3 signaling leads to the emergence of a self- autonomous, dominant subpopulation that accounts for the pathogenic activity of DEN FAPs. The specific Aims will test the hypothesis that STAT3-mediated activation of IL6 super-enhancers is a key event for the establishing a pathogenic feedforward IL6-STAT3 loop. In Aim 1 (3D regulation of IL6 locus in DEN FAPs), we will use chromosome conformation capture (3C)- and ChIP-based technologies to identify and characterize IL6 gene-interacting enhancers in DEN FAPs. In Aim 2 (Detection of DEN subFAPs and their and functional characterization), we will exploit single cell RNAseq to detect DEN subFAPs and establish a functional relationship between IL6-STAT3 hyperactivation and their self-autonomous expansion. In Aim 3 Pharmacological targeting of STAT3-activated super-enhancers in DEN FAPs, we will use pharmacological inhibition of STAT3-activated super-enhancers to determine whether interruption of IL6- STAT3 feedforward loop prevents the pathogenic potential of DEN subFAPs.